1. Technical Field
The present invention relates to a method for cell-free protein synthesis in which pH is controlled by using an enzyme. More specifically, the present invention relates to a method for cell-free protein synthesis which can provide, by controlling a decrease in pH using an amino acid decarboxylase, a cell-free protein in an enhanced synthesis amount, and can be used for screening of an active type protein without separating or purifying a synthesized protein.
2. Background Art
The cell-free protein synthesis generally indicates a technique for producing a large amount of a target protein within a short period of time in which only the intracellular protein synthesis mechanism involved with cellular protein synthesis and the factors of the mechanism are selected and only the protein synthesis process is artificially repeated outside a cell while the physiological regulation mechanism of a cell is excluded. With regard to the components required for biosynthesis of a protein, i.e., ribosome, initiation factor, elongation factor, termination factor, aminoacyl tRNA synthase, or the like, those included in a cell lysate can be used, or they can be separately added and used (Yoshihiro Shimizu et. al, 2001, Nature Biotechnology, 19(8): 751-755; Tae-Wan Kim et. al, 2006, Journal of Biotechnology, 126(4): 554-561).
Because continuous supply of ATP is required for a transcription reaction and a translation reaction of DNA by the components for protein synthesis, it is necessary for a solution for cell-free protein synthesis to have a mechanism for ATP regeneration. In this regard, it is noted that materials like acetyl phosphate, creatine phosphate, or phosphoenol pyruvate (PEP) that are conventionally used for ATP regeneration have a problem that they have not only poor ATP regeneration efficiency but also poor protein synthesis efficiency due to accumulation of inorganic phosphate.
To solve such problem, it is recently reported by Swartz, et. al. that, by using glucose as a material for ATP regeneration required for cell-free protein synthesis, smooth supply of ATP for a long period of time can be achieved, thus making it possible to have cell-free protein synthesis with high productivity, and it was also shown that, by using an inexpensive energy source like glucose for ATP regeneration, the productivity and economic efficiency of cell-free protein synthesis can be significantly enhanced (Kara A. Calhoun et. al, 2005, Biotechnology Progress, 21(4):1146-1153). As it undergoes a catabolic reaction and an oxidative phosphorylation process that are caused by enzymes present in a cell extract, glucose can regenerate several ATP molecules. Accordingly, it can be used as an efficient energy source in a system for cell-free protein synthesis.
However, when glucose and intermediates of glycolysis other than glucose are used for the reaction of cell-free protein synthesis, a decrease in pH of the reaction solution is caused in accordance with accumulation of organic acids that are derived from the glucose and intermediates. Such decrease in pH becomes a factor for limiting the productivity of cell-free protein synthesis as the activity of protein synthesis mechanism is inhibited by pH decrease (Ho-Cheol Kim et. al., 2011, Process Biochemistry, 46(6): 1366-1369). As such, it is essential to control the pH to be in a range in which the components of protein synthesis mechanism in a cell extract are not affected by it.
To have pH control, use of a chemical pH buffer agent like Tris and HEPES is generally carried out. However, pH decrease in a reaction solution caused by use of glucose or the like is not sufficiently inhibited by a pH buffer agent at the concentration which is generally used at present moment, and there is also a problem that, because the use of a buffer agent at high concentration is accompanied with a corresponding increase in salt concentration, it may become a factor which can inhibit the protein synthesis.
Furthermore, because the buffer solution used for pH control cannot be removed after the reaction of cell-free protein synthesis, if the activity of an enzyme synthesized by cell-free protein synthesis is to be analyzed in view of pH, a change in pH caused by the enzyme activity is suppressed due to the inclusion of the buffer solution of a solution for synthesis in a solution for activity analysis, and thus an accurate analysis of enzyme activity cannot be made.
Meanwhile, in Korean Patent Registration No. 0749053, a method for cell-free protein synthesis is disclosed, and in Korean Patent Registration No. 0733712, production of a cell extract for cell-free protein synthesis and a method for protein synthesis using it are disclosed. Furthermore, in Korean Patent Registration No. 1229849, a method for enhancing protein expression yield of cell-free protein synthesis according to addition of an anti-foaming agent is disclosed. However, a technique for solving the aforementioned problems has not been suggested.